RESUMO
Streptococcus pyogenes (group A streptococcus [GAS]) is a human-specific pathogen that causes a variety of diseases ranging from superficial infections to life-threatening diseases. SpeB, a potent extracellular cysteine proteinase, plays an important role in the pathogenesis of GAS infections. Previous studies show that SpeB expression and activity are controlled at the transcriptional and posttranslational levels, though it had been unclear whether speB was also regulated at the posttranscriptional level. In this study, we examined the growth phase-dependent speB mRNA level and decay using quantitative reverse transcription-PCR (qRT-PCR) and Northern blot analyses. We observed that speB mRNA accumulated rapidly during exponential growth, which occurred concomitantly with an increase in speB mRNA stability. A closer observation revealed that the increased speB mRNA stability was mainly due to progressive acidification. Inactivation of RNase Y, a recently identified endoribonuclease, revealed a role in processing and degradation of speB mRNA. We conclude that the increased speB mRNA stability contributes to the rapid accumulation of speB transcript during growth.
Assuntos
Proteínas de Bactérias/biossíntese , Exotoxinas/biossíntese , Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , RNA Mensageiro/biossíntese , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética , Proteínas de Bactérias/genética , Northern Blotting , Exotoxinas/genética , Perfilação da Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this investigation, we identify the CodY protein from Streptococcus pyogenes as a pleiotropic transcription regulator with global features. The notion that acquisition of nutrients by this polyauxotrophic organism is the primary event occurring during the establishment of infection and that virulence expression is a result of this quest, led us to study the action of codY and relA genes on transcriptional gene expression under different nutritional conditions using complex and chemically defined media. Real-time reverse transcription PCR was used to determine the extent to which inactivation of codY and relA affects the mRNA levels of selected transcription factors, virulence genes, transporters, and genes encoding metabolic enzymes. The results show that CodY and RelA did not affect the expression of each other but that both exhibited strong negative autoregulatory properties. Genes negatively controlled by the relA-directed stringent response to amino acid starvation included, besides relA itself, transporters, metabolic enzymes, and at least two virulence genes (graB and speH). The expression of many genes of all four groups studied proved to be subject to direct or indirect control by CodY, often in a nutritional status-dependent fashion. One of the most important results implicates CodY in growth phase-dependent positive transcriptional regulation of pel/sagA and mga, loci that themselves positively affect the expression of numerous virulence factors. Increasing the cellular activity of nicotinamidase in both a codY mutant and wild-type background induced extensive transcriptional reprogramming, altering, among others, the growth phase-dependent transcription pattern of the genes for cysteine protease (speB) and several transporters. Inasmuch as CodY influenced the expression of other regulators (pel/sagA, mga, covRS, ropB, pyrR), its action is amplified and expands the complex regulatory network that governs gene expression in S. pyogenes.
Assuntos
Regulação Bacteriana da Expressão Gênica , Ligases/fisiologia , Proteínas Repressoras/fisiologia , Streptococcus pyogenes/genética , Adaptação Fisiológica , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Northern Blotting , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Enzimas/biossíntese , Enzimas/genética , Deleção de Genes , Ligases/genética , RNA Bacteriano/análise , RNA Mensageiro/análise , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Virulência/biossíntese , Fatores de Virulência/genéticaRESUMO
A subtraction library of group B streptococcus (GBS) strain O9OR with GAS chromosomal DNA (strain SF370) was constructed and more than 100 plasmid clones sequenced. DNA sequences of the plasmid inserts were analyzed using the BLAST gene search. Most inserts had little or no homology to GAS chromosomal DNA and 26 clones from the library had no gene homologues in the gene bank. The majority of genes discovered represented house keeping GBS genes, but several could be considered as possible virulence factors. Inserts from 21 clones were labeled and used as probes for hybridization with GBS DNA fragments separated by pulsed field electrophoresis. A genetic map of GBS strain O9OR was constructed.